Microbes are a common cause of illness and death. In order for medical professionals to diagnose and treat an illness it is often necessary to identify the disease causing agent. Moreover, identifying the cause of an outbreak of disease allows steps to be taken to prevent the spread of the disease.
Microbes are sometime identified based on physical characteristics such as size, shape, colony type, and staining characteristics. While such physical characteristics can be used to eliminate some of the possible disease causing agents, it is often not possible to identify a microbe based on these characteristics alone. For example many species of bacteria are further classified into strains. Some members of the species can be harmless or even beneficial microbes, while others may be pathogenic.
Because the disease causing strains have a gene or genes that render them pathogenic, DNA analysis can be used to identify these strains. The DNA analysis can provide a positive identity of a pathogenic organism. However, the amount of information provided is limited, and the process can take a considerable amount of time. When the purpose of identifying the microbe is to treat a disease or prevent its spread the DNA analysis may take too long to be useful.
Amplified Fragment Length Polymorphism (AFLP) and strain-specific polymerase chain reaction (PCR) analyses are the methods used for determining the identity of microbes. These procedures provide significantly more information than standard DNA analysis and are more rapid and less expensive than extensive DNA sequencing. AFLP analysis can be used for rapidly characterizing unknown pathogenic species and strains, thereby providing valuable information for developing a therapeutic response to an outbreak thereof Strain-specific PCR analysis can rapidly identify a previously studied threat species, even if the sample is present in a complex sample mixture. Both methods require further analysis of the reaction results to determine the size of the DNA fragments. Simple detection of the presence or absence of the fragment is insufficient. The time required to complete the reaction analysis is presently between 15 and 20 minutes. However, size analysis is currently conducted using an automated DNA sequencer. The gel procedure requires approximately three hours to complete, plus additional time to set up the sequencing unit and download the resulting data. When faced with the potential or actual release of a biothreat agent, it is important to obtain the genetic information about the released organism in a significantly shorter time period. It is also important to be able to conduct analyses in a field laboratory using affordable apparatus. Flow cytometry analysis does not allow resolution of such small fragments. Commercial capillary electrophoresis sequencers have sufficient resolution, but have a high cost and are not suitable for rapid fragment size determination.
Currently, DNA size analysis requires approximately three hours to run on a gel, plus additional time to set up the sequencing unit and download the data. Flow cytometry analysis does not resolve 100 to 500 base pair (bp) fragments with 1 bp resolution which is required. Commercial capillary electrophoresis sequencers that can resolve 1 bp are costly and do not permit size determination.
Capillary electrophoresis is a powerful analytical technique that can be used to separate and detect a number of analytes according to their charge. Traditionally, electrophoresis has been practiced on slab gels. However, the use of capillaries introduces a number of advantages including small sample and electrolyte volumes, high efficiency separations, and small instrument size. The capillary systems are also amenable to miniaturization.
In capillary electrophoresis, an electrical field is applied along the length of a fused silica capillary filled with a buffer. Typically, particle samples are introduced at the anode and are carried towards the cathode by the electroosmotic movement of the electrolyte and the electrophoretic movement of the analyte ions. The sample is separated according to the charge to size ratio of the analyte ions.
The separated sample is detected at some optimal fixed point in the capillary. A number of detection schemes are used in capillary electrophoresis including spectroscopic techniques such as UV-Vis absorbance, fluorescence, and raman; mass spectrometry; and electrochemical detection. Separations can be performed on analyte samples including charged particles such as small organic acids and bases, proteins, peptides, amino acids, and nucleic acids. Neutral molecules can also be separated with a special form of capillary electrophoresis called micellar electrokinetic chromatography. Because the entire sample must migrate past the detection window, the capillary electrophoretic systems tend to be long and require significant time for a good separation of particles.
In light of the foregoing, it would be a significant advancement in the art to provide a device that could rapidly separate and detect particles in a sample solution. It would be a further advancement if the device could be produced inexpensively. It would be a further advancement if the device were amenable to a high throughput system and miniaturization. It would be a further advancement if the device used capillary electrophoresis. Such a device is disclosed and claimed herein.